g-Aminobutyric Acid (GABA) is the major inhibitory neurotransmitter in the mammalian brain, and plays a prominent inhibitory role in the brainstem and spinal cord as well. One mechanism through which GABA produces its inhibitory action is via GABAA receptors which produce fast synaptic inhibition of neurons by activation of intrinsic CI- channels. GABA opening of CI- channels produces an inward movement of CI-, driven by a low intracellular CI- concentration which is maintained by an active CI- extrusion mechanism: presumed to be the neuronal-specific isoform of the K-CI cotransporter, KCC2. We have disrupted Sic12a5, the gene encoding this isoform of the K-CI cotransporter, and the homozygous mutant mice die shortly after birth of repeated seizures. Epileptic seizure activity in the KCC2 knockout brain suggests hyper-excitability, in agreement with the putative role of KCC2 in controlling hyperpolarizing GABA responses. This proposal is aimed at understanding the role of KCC2 in controlling CNS excitability and epilepsy. We will 1) investigate the developmental role of KCC2 in regulating intracellular CI- and controlling the maturation of GABA hyperpolarization, 2) investigate the role of KCC2 in preventing hyper-excitability and the participation of the cotransporter in depolarizing GABA responses during high frequency synaptic activation. This will be achieved through detailed electrophysiological measurements in hippocampal slices and isolated cortical neurons, 3) develop brain-region-specific and inducible knockout of KCC2 to study the knockout phenotype in the adult and examine the effect of graded reduction in KCC2 expression, and 4) examine the determinants of KCC2 function and regulation, by focusing mainly on phosphorylation/dephosphorylation of the protein. The epileptic seizure phenotype of the KCC2 knockout mouse demonstrates the importance of KCC2 in preventing hyper-excitability and controlling CNS function. Results of these molecular, physiological, and behavioral studies will lead to a better understanding of the relationship between cation-chloride cotransporters, ion homeostasis, synaptic transmission and brain excitability.